Chemical characterization (LC-MS-ESI), cytotoxic activity and intracellular localization of PAMAM G4 in leukemia cells.

Generation 4 of polyamidoamine dendrimer (G4-PAMAM) has several biological effects due to its tridimensional globular structure, repetitive branched amides, tertiary amines, and amino-terminal subunit groups liked to a common core. G4-PAMAM is cytotoxic due to its positive charges. However, its cytotoxicity could increase in cancer cells due to the excessive intracellular negative charges in these cells. Furthermore, this work reports G4-PAMAM chemical structural characterization using UHPLC-QTOF-MS/MS (LC-MS) by electrospray ionization to measure its population according to its positive charges. Additionally, the antiproliferative effects and intracellular localization were explored in the HMC-1 and K-562 cell lines by confocal microscopy. The LC-MS results show that G4-PAMAM generated multivalent mass spectrum values, and its protonated terminal amino groups produced numerous positive charges, which allowed us to determine its exact mass despite having a high molecular weight. Additionally, G4-PAMAM showed antiproliferative activity in the HMC-1 tumor cell line after 24 h (IC50 = 16.97 µM), 48 h (IC50 = 7.02 µM) and 72 h (IC50 = 5.98 µM) and in the K-562 cell line after 24 h (IC50 = 15.14 µM), 48 h (IC50 = 14.18 µM) and 72 h (IC50 = 9.91 µM). Finally, our results showed that the G4-PAMAM dendrimers were located in the cytoplasm and nucleus in both tumor cell lines studied.

Molecular modeling and LC-MS-based metabolomics of a glutamine-valproic acid (Gln-VPA) derivative on HeLa cells.

Glutaminase plays an important role in carcinogenesis and cancer cell growth. This biological target is interesting against cancer cells. Therefore, in this work, in silico [docking and molecular dynamics (MD) simulations] and in vitro methods (antiproliferative and LC-MS metabolomics) were employed to assay a hybrid compound derived from glutamine and valproic acid (Gln-VPA), which was compared with 6-diazo-5-oxo-L-norleucine (DON, a glutaminase inhibitor) and VPA (contained in Gln-VPA structure). Docking results from some snapshots retrieved from MD simulations show that glutaminase recognized Gln-VPA and DON. Additionally, Gln-VPA showed antiproliferative effects in HeLa cells and inhibited glutaminase activity. Finally, the LC-MS-based metabolomics studies on HeLa cells treated with either Gln-VPA (IC60 = 8 mM) or DON (IC50 = 3.5 mM) show different metabolomics behaviors, suggesting that they modulate different biological targets of the cell death mechanism. In conclusion, Gln-VPA is capable of interfering with more than one pharmacological target of cancer, making it an interesting drug that can be used to avoid multitherapy of classic anticancer drugs.

In silico search, chemical characterization and immunogenic evaluation of amino-terminated G4-PAMAM-HIV peptide complexes using three-dimensional models of the HIV-1 gp120 protein.

Peptide epitopes have been widely used to develop synthetic vaccines and immunotherapies. However, peptide epitopes may exhibit poor absorption or immunogenicity due to their low molecular weights. Conversely, fourth-generation polyamidoamine (G4-PAMAM) dendrimers are nonimmunogenic and relatively nontoxic synthetic nanoparticles that have been used as adjuvants and nanocarriers of small peptides and to improve nasal absorption. Based on this information, we hypothesized that the combination of intranasal immunization and G4-PAMAM dendrimers would be useful for enhancing the antibody responses of HIV-1 gp120 peptide epitopes. Therefore, we first used structural data, peptide epitope predictors and docking and MD simulations on MHC-II to identify two peptide epitopes on the CD4 binding site of HIV-1 gp120. The formation of G4-PAMAM-peptide complexes was evaluated in silico (molecular docking studies using different G4-PAMAM conformations retrieved from MD simulations as well as the MMGBSA approach) and validated experimentally (electrophoresis, 1H NMR and cryo-TEM). Next, the G4-PAMAM dendrimer-peptide complexes were administered intranasally to groups of female BALB/cJ mice. The results showed that both peptides were immunogenic at the systemic and mucosal levels (nasal and vaginal), and G4-PAMAM dendrimer-peptide complexes improved IgG and IgA responses in serum and nasal washes. Thus, G4-PAMAM dendrimers have potential for use as adjuvants and nanocarriers of peptides.

Computational Study of the Binding Modes of Diverse DPN Analogues on Estrogen Receptors (ER) and the Biological Evaluation of a New Potential Antiestrogenic Ligand.

Estrogen (17ß-estradiol) is essential for normal growth and differentiation in the mammary gland. In the last three decades, previous investigations have revealed that Estrogen Receptor Alpha (ERα) plays a critical role in breast cancer. More recently, observations regarding the widespread expression of ERß-like proteins in normal and neoplastic mammary tissues have suggested that ERß is also involved in the mentioned pathology. Design of new drugs both steroidal and nonsteroidal that target any of these receptors represents a promise to treat breast cancer although it remains a challenge due to the sequence similarity between their catalytic domains. In this work, we propose a new set of compounds that could effectively target the estrogen receptors ERα and ERß. These ligands were designed based on the chemical structure of the ERß-selective agonist Diarylpropionitrile (DPN). The designed ligands were submitted to in silico ADMET studies, yielding in a filtered list of ligands that showed better drug-like properties. Molecular dynamics simulations of both estrogen receptors and docking analysis were carried-out employing the designed compounds, from which two were chosen due to their promising characteristics retrieved from theoretical results (docking analysis or targeting receptor predictions). They were chemically synthetized and during the process, two precursor ligands were also obtained. These four ligands were subjected to biological studies from which it could be detected that compound mol60b dislplayed inhibitory activity and its ability to activate the transcription via an estrogenic mechanism of action was also determined. Interestinly, this observation can be related to theoretical binding free energy calculations, where the complex: ERß-mol60b showed the highest energy ΔGbind value in comparison to others.

Current Status and Future Prospects to Achieve Foot-and-Mouth Disease Eradication in South America.

South America has a favourable position with respect to foot-and-mouth disease (FMD) compared with other FMD-affected regions due to the elimination of endemic clinical presentation of the disease. South America has reached the final stage of control and aims to eradicate the disease in the region under the provisions of the Hemispheric Program for the Eradication of FMD 2011-2020 (PHEFA). This programme aims at bringing eradication to completion, thereby eliminating the pool of foot-and-mouth disease genotypes active in South America. This plan includes a regional political agreement that provides strategies and technical guidelines for the eradication of foot-and-mouth disease from South America. It incorporates knowledge and experience regarding the disease's history and its connection with the different production systems, animal movement and trade. The Pan American Foot and Mouth Disease Center has led the control and eradication programmes, providing the framework for designing national and subregional programmes that have led to significant progress in controlling the disease in South America. The current situation is the result of several factors, including the proper implementation of a national control programmes, good veterinary infrastructure in most countries and public-private participation in the process of eradicating the disease. Notwithstanding the favourable health status, there are significant challenges for the goal of eradication. At this stage, South American countries should enhance their surveillance strategies particularly through the use of target or risk-based surveys that contribute to increase the degree of sensitivity in the search for viral circulation in the context of absence of clinical occurrence of FMD.

Deciphering the GPER/GPR30-agonist and antagonists interactions using molecular modeling studies, molecular dynamics, and docking simulations.

The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30's molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π-π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.